This application claims priority of Japanese Patent Application No. 2001-82819, filed Mar. 22, 2001.
The present invention relates to a method for obtaining a malignant cell line, to a cell line obtainable by the method, to a method for screening for an angiogenesis inhibitor and an anticancer agent by using the obtained cell line, and to an angiogenesis inhibitor and an anticancer agent obtainable by the screening method.
In recent years, many reports have been made on the close relationship between the malignancy of a cancer (hematogenous metastasis, lymphogenous metastasis, dissemination, retention of cancerous ascites or pleural effusion, cancerous cachexia, and shortening of survival time of a host due to them) and angiogenesis (Macchiarini P, Fontanini G, Hardin M J, Squartini F, Angeletti C A, Lancet, 1992, Vol. 340, P145-146; Horak ER, Leek R, Klemk N, Lejeune S, Smith K, Stuart N, Greenall M, Steoniewska K, Harris A N, Lancet, 1992, Vol. 340, P1120-1124; Takahashi Y, Bucana CD, Cleary KR, Ellis LM, Int. J. Cancer, 1998, Vol. 79, P34-38). It has been reported that angiogenesis is induced by various angiogenic factors; and that among these factors, expressions of vascular endothelial growth factor (VEGF), platelet-derived growth factor, hepatocyte growth factor and fibroblast growth factor correlate with the prognosis of patients suffering from cancers. These suggest that angiogenesis induced by an angiogenic factor plays a key role in the malignant alternation of cancers.
Therefore, a model for evaluating the malignant alternation of cancers by an angiogenic factor can be a useful tool for the development of therapeutic agents for malignant tumors. Also, it can be a useful tool for the development of therapeutic agents for angiogenic diseases in which such angiogenic factors are involved.
Hitherto, the present inventors have reported on a model for directly evaluating VEGF-dependent hematogenous metastasis by introducing VEGF, which is one of angiogenic factors, into human colon carcinoma WiDr cells to increase the ability of metastasis (Funahashi et al., Jpn. J. Cancer Res., Vol. 90 Supplement, P132, 1999). However, since VEGF is not merely an angiogenic factor but also a vascular permeability factor and since WiDr cells conventionally have high angiogenesis-inducing ability, it is difficult to directly evaluate the introduced VEGF-dependent angiogenesis, and involvement of VEGF-dependent angiogenesis in the malignant alternation of cancers has not been elucidated yet.
As for cancers, while angiogenesis is a major factor for future aggravation, it is considered that many kinds of angiogenic factors are involved in angiogenesis induced by cancers.
In order to develop an effective anticancer agent that exhibits its effect by inhibition of angiogenesis, it must exhibit its effect not only on angiogenesis induced by VEGF but also on angiogenesis induced by various kinds of factors, and hence it is considered that evaluation of angiogenesis dependent on various angiogenic factors, in particular, evaluation of the effects on the malignant alternation of cancers based on the imparted angiogenic ability in respective organs is important. However, with conventional models, it has been difficult to sufficiently evaluate angiogenesis for an individual angiogenic factor.
Therefore, a main object of the present invention is to procure a cell which has acquired malignancy (hematogenous metastasis, lymphogenous metastasis, dissemination, retention of cancerous ascites or pleural effusion, cancerous cachexia, and shortening of survival time of a host due to them) as a result of increase in angiogenesis-inducing ability due to expression of an angiogenic factor, and further to establish a system for screening for an angiogenesis inhibitor and an anticancer agent by use of the cell.
The present inventors have introduced a gene of an angiogenic factor into a cell line having low in vivo angiogenesis-inducing ability to create a cell line having high angiogenic factor productivity. In addition, they have tried to create a model for evaluating the in vivo angiogenesis and the malignant alternation of cancers by use of the cell line having high angiogenic factor productivity.
As a result, it has been found that in the cell line having high angiogenic factor productivity, the in vivo angiogenesis ability is increased and further, increase in the ability of growth in a region of transplantation, increase in lymph node metastasis, retention of ascites, and shortening of survival time which is considered to be due to overall influences of these have been observed. Thus, it has been revealed that the introduced angiogenic factor alone induces angiogenesis and the malignant alternation of cancers.
Also, it has been revealed that cell lines having high angiogenic factor productivity differ in characteristics, dependent on the kind of angiogenic factors to be introduced, and they can serve as a model for evaluating the malignant alternation of cancers reflecting in vivo angiogenesis differing in the property depending on the variety of angiogenic factors and malignant tumors dependent on various angiogenic factors in clinical practices, so that they provide a useful tool for the development of therapeutic agents for malignant tumors and angiogenic diseases in clinical practices.
Based on the above findings, the present inventors have completed the present invention.
That is, the present invention provides the following.
(1) A method for producing a cell to which angiogenesis-inducing ability has been imparted, comprising the steps of an introducing a gene of angiogenic factor into a human or animal cell having low angiogenesis-inducing ability, and selecting a cell that overexpresses the angiogenic factor.
(2) The method according to (1), wherein the cell having low angiogenesis-inducing ability is human pancreatic cancer KP-1 cell.
(3) A method for screening for a substance having angiogenesis inhibitory activity, comprising the steps of bringing a candidate substance into contact with a cell obtained by the method as defined in (1) or (2), and selecting a substance having activity of inhibiting the angiogenesis-inducing ability of the cell.
(4) A method for producing an angiogenesis inhibitory composition, comprising the steps of obtaining a substance having angiogenesis inhibitory activity by the method as defined in (3), and preparing the composition from the obtained substance and a carrier.
(5) A method for screening for a substance having anticancer activity, comprising the steps of bringing a candidate substance into contact with a cell obtained by the method as defined in (1) or (2), and selecting a substance having anticancer activity on the cell.
(6) A method for producing an anticancer composition, comprising the steps of obtaining a substance having anticancer activity by the method as defined in (5), and preparing the composition from the obtained substance and a carrier.
According to the present invention, it has been elucidated that overexpression of angiogenic factors in less malignant cells, preferably KP-1 cells, results in increase in angiogenic ability and malignant alternation, and hence the present invention provides cells useful for examining the effect of angiogenic factors and enables screening for an angiogenesis inhibitor and an anticancer agent by use of such cells.